Cytotoxic T-cell epitopes of the papillomavirus L1-protein and use thereof in diagnostics and therapy

ABSTRACT

The present invention relates to a papillomavirus T-cell epitope having an amino acid sequence AQIFNKPYW, AGVDNRECI and/or to a functionally active variant thereof, also to their use in diagnostics and therapy.

This application is the U.S. National Stage of International ApplicationNo. PCT/EP2000/05005, filed May 31, 2000, which claims the benefit ofGerman Application No. 19925235.1, filed Jun. 1, 1999, all of which arehereby incorporated by reference.

The present invention relates to a papillomavirus T-cell epitope havingan amino acid sequence AQIFNKPYW, AGVDNRECI and/or to a functionallyactive variant thereof, and also to its use in diagnostics and therapy.

The papillomaviruses, also called wart viruses, are double-stranded DNAviruses with a genome size of about 8000 base pairs and an icosahedralcapsid of approx. 55 nm in diameter. Up until now, more than 100different human-pathogenic papillomavirus types (HPV) are known, some ofwhich, for example HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-45,HPV-52 or HPV-58, may cause malignant tumors and others, for exampleHPV-6, HPV-11 or HPV-42, may cause benign tumors.

The papillomavirus genome can be divided into three parts: the firstpart relates to a noncoding region containing regulatory elements forvirus transcription and replication. The second region, the “E” (early)region, contains various protein-encoding sections E1-E7 of which, forexample, the E6 and E7 proteins are responsible for transformation ofepithelial cells and the E1 protein controls the DNA copy number. The E6and E7 regions are “oncogenes” which are also expressed in malignantlydegenerate cells. The third region, also called L (late) region,contains two protein-encoding sections L1 and L2 which code forstructural components of the virus capsid. Over 90% of the L protein ispresent in the viral capsid, the L1:L2 ratio generally being 30:1. Inaccordance with the present invention, the term L1 protein means themain capsid protein of papillomaviruses (Baker T. et al. (1991) Biophys.J. 60, 1445).

In over 50% of cases, HPV-16 is connected with cervical cancer(carcinoma of the cervix). HPV-16 is the main risk factor for theformation of cervical neoplasms. The immune system plays an importantpart in the progress of the disease. Thus, cellular immune responses andin particular antigen-specific T lymphocytes are presumably importantfor the defense mechanism. It has furthermore been found that inhigh-grade malignant cervical intraepithelial neoplasms (CIN II/III) andcervical tumors the E7 gene is expressed constitutively in all layers ofthe infected epithelium. The E7 protein in particular is thereforeconsidered as a potential tumor antigen and as a target molecule foractivated T cells (see, for example, WO 93/20844). The E7-inducedcellular immune response in the patient, however, is apparently notstrong enough to influence the course of the disease. The immuneresponse may possibly be amplified by suitable vaccines.

It has been possible to show that expression of the L1 gene and/orcoexpression of the L1 and L2 genes can lead to the formation ofcapsomers, stable capsomers, capsids or virus-like particles (VLPs)(see, for example, WO 93/02184, WO 94/20137 or WO 94/05792). Capsomersmean an oligomeric configuration which is composed of five L1 proteins.The capsomer is the basic building block of which viral capsids arecomposed. Stable capsomers mean capsomers which are incapable ofassembling to form capsids. Capsids mean the papillomavirus coat whichis, for example, composed of 72 capsomers (Baker T. et al. (1991)Biophys. J. 60, 1445). VLP means a capsid which is morphologically andin its antigenicity identical to an intact virus. It was possible to usethe VLPs in various animal systems for causing a humoral immune responsecharacterized by the formation of neutralizing antibodies. The formationof virus-neutralizing antibodies against L1 and/or L2 protein, however,is of relatively low clinical importance if the virus infection hasalready taken place, since for the elimination of virus-infected cells avirus-specific cytotoxic T-cell (CTL) response rather than antibodiesseems to be necessary. And, although VLPs are capable of causing acytotoxic T-cell response, an immune response exclusively directedagainst the capsid proteins L1 and/or L2 appears unsuitable forcontrolling a tumor caused by papillomaviruses.

Therefore, “chimeric papillomavirus-like particles” (CVLPs) whichcomprise a fusion protein of the capsid protein L1 and the potentialtumor antigen E7 (WO 96/11272 and Muller, M. et al. (1997) Virology,234, 93) have been developed. The CVLPs caused only to a small extent ahumoral immune response directed against the E7 protein (Muller, M. etal. (1997), supra). Some of the CVLPs tested, however, do indeed inducethe desired E7-specific cytotoxic T-cell response in mice (see also PengS. et al. (1998) Virology 240, 147-57). As a result, CVLPs are ofinterest both for the development of a vaccine and for the treatment ofalready established infections and tumors resulting therefrom, since theE7 tumor cell peptides presented via MHC molecules of class I wouldrepresent target molecules of cytotoxic T cells.

A vaccine comprising CVLPs is based on the principle of the CVLPspseudo-infecting cells. This means that CVLPs and viruses alike get intothe cell, are processed there to peptides, and the peptides are loadedonto MHC class I and II molecules and finally presented to CD8- orCD4-positive T cells. As a consequence of this stimulation, CD8 cellsmay differentiate into cytotoxic T cells and then cause a cellularimmune response, whereas CD4 cells develop into T helper cells andstimulate B cells to give a humoral or CD8-positive T cells to give acytotoxic immune response and may themselves induce lysis of infectedcells.

Small peptides may bind to MHC class I molecules already on the cellsurface and then stimulate without further processing CD8- orCD4-positive cells to give a cellular immune response. However, aparticular peptide can be bound only by particular MHC molecules. Due tothe large polymorphism of MHC molecules in natural populations, aparticular peptide can therefore be bound and presented only by a smallpart of a population. In accordance with the present invention,presentation means binding of a peptide or protein fragment to an MHCmolecule, it being possible for said binding to take place, for example,in the endoplasmic reticulum, the extracellular space, the endosomes,proendosomes, lysosomes or protysosomes, and said MHC molecule-peptidecomplex then being bound on the extracellular side of the cell membraneso that it can be recognized specifically by immune cells.

Since CVLPs cause both a cellular and a humoral immune response and arenot MHC-restricted, this technology is generally suitable for thedevelopment of vaccines, since an L1 portion provides the ability toform particles and an additional antigen portion is fused to said L1portion.

For the development of CVLPs of this kind it is absolutely necessary tohave a functional assay system available which can be used to studydirectly the immunogenicity of CVLPs. Such an assay system should havethe property that CVLPs with different antigen proportions can bestudied by using the same assay system. Since the cellular immuneresponse is of crucial importance for immunological therapies of tumorsor viral diseases, the object arose to make it possible to measure thecellular immune response caused by CVLPs.

This object was achieved by identifying T-cell epitopes which inconnection with MHC molecules, and in a particular embodiment with(H2-D^(b)) MHC molecules, cause, for example, a cytotoxic T-cellresponse in vivo and in vitro. Said peptides preferably have thesequence AQIFNKPYW or AGVDNRECI. These sequences are part of the L1peptide of HPV16. They include the amino acid regions 330 to 338(L1₃₃₀₋₃₃₈) and 165 to 173 (L1₁₆₅₋₁₇₃).

The present invention therefore relates to a T-cell epitope having anamino acid sequence AQIFNKPYW, AGVDNRECI and/or to a functionally activevariant thereof.

A functionally active variant of AQIFNKPYW (SEQ ID NO: 1) or AGVDNRECI(SEQ ID NO: 2) means a T-cell epitope which, in a T-cell cytotoxicityassay system (see, for example, Examples 2-5 of the present invention),has a cytotoxicity which, compared to the cytotoxicity of AQIFNKPYW (SEQID NO: 1) or AGVDNRECI (SEQ ID NO: 2), corresponds to at least the sumof the average of the negative controls and three times the standarddeviation, preferably of at least approx. 30%, in particular at leastapprox. 50% and particularly preferably of at least approx. 80%.

An example of a preferred variant is a T-cell epitope having a sequencehomology to AQIFNKPYW (SEQ ID NO: 1) or AGVDNRECI (SEQ ID NO: 2) of atleast approx. 65%, preferably at least approx. 75% and in particular atleast approx. 85% at the amino acid level. Other preferred variants arealso T-cell epitopes which are structurally homologous to AQIFNKPYW (SEQID NO: 1) or AGVDNRECI (SEQ ID NO: 2). Such epitopes may be found bygenerating specific T-cells against the T-cell epitopes AQIFNKPYW (SEQID NO: 1), AGVDNRECI (SEQ ID NO: 2) (DeBruijn M. L. et al. (1991) Eur.J. Immunol. 21, 2963-70; and DeBruijn M. L. (1992) Eur. J. Immunol. 22,3013-20) and assaying, for example, synthetically produced peptides ofchoice for recognition by the peptide-specific T cells (see examples).The T-cell epitopes in particular mean cytotoxic T-cell epitopes.However, noncytotoxic T cells are also known which can likewiserecognize MHC I molecules so that the present invention also includesnoncytotoxic T-cell epitopes as variant.

Another embodiment of the present invention is a T-cell epitope which ispart of a compound, the compound not being a naturally occurring L1protein of a papillomavirus and not being an exclusively N-terminal orexclusively C-terminal deletion mutant of a naturally occurring L1protein of a papillomavirus. In a particular embodiment, a T-cellepitope having an amino acid sequence AQIFNKPYW (SEQ ID NO: 1),AGVDNRECI (SEQ ID NO: 2), and/or a functionally active variant may becontained in an L1 protein of a different papillomavirus or in achimeric L1 protein, for example an HPV18L1E7 fusion protein. Such acompound of the invention may have the ability to form CVLPs.

As part of a compound, said T-cell epitope may preferably be apolypeptide which preferably contains further amino acid sequences, andin particular a fusion protein. In particular, the compound may be apolypeptide of at least approx. 50 amino acids, preferably of at leastapprox. 35 amino acids, in particular of at least approx. 20 amino acidsand particularly preferably of at least approx. 9 amino acids, inlength.

In order to detect the compound or to modify its T-cell bindingactivity, said compound may contain a chemical, radioactive isotope,nonradioactive isotope and/or fluorescent label of the T-cell epitopeand/or of said fusion protein.

Examples of chemical substances known to the skilled worker, which aresuitable for chemical labeling according to the invention, are: biotin,FITC (fluorescein isothiocyanate) or streptavidin.

In a possible embodiment a peptide is modified such that it contains atleast one lysine. In a manner known to the skilled worker biotin or FITC(fluorescein isothiocyanate) is coupled to said lysine. A peptidemodified in this way is bound to an appropriate MHC molecule or to acell containing appropriate MHC molecules. The peptide may then bedetected via labeled avidin or streptavidin or directly via FITCfluorescence.

Examples of isotopes known to the skilled worker, which are suitable forradioactive isotope labeling according to the invention are: ³H, ¹²⁵I,¹³¹I, ³²P, ³³P or ¹⁴C.

Examples of isotopes known to the skilled worker, which are suitable fornonradioactive isotope labeling according to the invention are: ²H, or¹³C.

Examples of fluorescent substances known to the skilled worker, whichare suitable for fluorescence labeling according to the invention are:¹⁵²Eu, fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phtaldehyde or fluorescamine.

Further label not listed here, which may also be used for labeling inaccordance with this invention, are known to the skilled worker.

Examples of inventive chemical modifications known to the skilled workerare the transfer of acetyl, phosphate and/or monosaccharide groups.

Inventive polypeptides of approx. 50 amino acids in length may beprepared, for example, by chemical peptide synthesis. Longerpolypeptides are preferably generated by genetic engineering. Thepresent invention therefore further relates to a nucleic acid constructfor expressing said T-cell epitope or compounds containing the followingcomponents: (a) at least one regulatory element and (b) at least onenucleic acid coding for an amino acid sequence of the compound of theinvention. Said nucleic acid construct is preferably made of DNA or RNA.Suitable regulatory elements allow, for example, constitutive,regulatable, tissue-specific, cell cycle-specific or metabolicallyspecific expression in eukaryotic cells or constitutive, metabolicallyspecific or regulatable expression in prokaryotic cells. Regulatableelements according to the present invention are promoters, activatorsequences, enhancers, silencers, and/or repressor sequences.

Examples of suitable regulatable elements which make constitutiveexpression in eukaryotes possible are promoters recognized by RNApolymerase III or viral promoters such as CMV enhancer, CMV promoter,SV40 promoter and viral promoter and activator sequences derived, forexample, from HBV, HCV, HSV, HPV, EBV, HTLV or HIV.

Examples of regulatable elements which make regulatable expression ineukaryotes possible are the tetracyclin operator in combination with acorresponding repressor (Gossen M. et al (1994) Curr. Opin. Biotechnol.5, 516-20).

Examples of regulatable elements which make tissue-specific expressionin eukaryotes possible are promoters or activator sequences frompromoters or enhancers of those genes coding for proteins which areexpressed only in particular cell types.

Examples of regulatable elements which make cell cycle-specificexpression in eukaryotes possible are the promoters of the followinggenes: cdc25C, cyclin A, cyclin E, cdc2, E2F, B-myb or DHFR (Zwicker J.and Muller R. (1997) Trends Genet. 13, 3-6).

Examples of regulatable elements which make metabolically specificexpression in eukaryotes possible are promoters regulated by hypoxia, byglucose deficiency, by phosphate concentration or by heat shock.

In order to make it possible to introduce said nucleic acid and thusexpress the polypeptide in a eukaryotic or prokaryotic cell bytransfection, transformation or infection, the nucleic acid may bepresent as plasmid, or as part of a viral or nonviral vector. Thepresent invention therefore further relates to a vector, in particularan expression vector which contains a nucleic acid coding for apolypeptide of the invention. Viral vectors particularly suitable hereare: baculo viruses, vaccinia viruses, adenoviruses, adeno-associatedviruses and herpes viruses. Nonviral vectors particularly suitable hereare: virosomes, liposomes, cationic lipids or polylysine-conjugated DNA.

The present invention further relates to a cell containing, preferablypresenting, at least one T-cell epitope. In a particular embodiment, thecell is transfected, transformed or infected by one of the vectorsmentioned. This cell expresses the polypeptide of the invention underconditions known to a skilled worker which lead to activation of theregulatable elements used in each case. The polypeptide can then beisolated from said cell and purified, for example by using one of theabovementioned labels. Cells which are suitable for the preparation bygenetic engineering and subsequent purification of the expressedcompounds of the invention are prokaryotic and eukaryotic cells, inparticular bacteria cells such as, for example, E. coli, yeast cellssuch as, for example, S. cerevisiae, insect cells such as, for example,Spodoptera frugiperda cells (Sf-9) or Trichoplusia ni cells or mammaliancells such as, for example, COS cells or HeLa cells.

A particular embodiment is using the cell itself which expresses thepolypeptide of the invention, and, in a particularly preferredembodiment, the cell presents parts of the polypeptide of the inventionvia MHC-1 molecules on the cell surface. Suitable cells for preparingthe cell of the invention are antigen-presenting cells such as, forexample, B cells, macrophages, dendritic cells, embryonic cells orfibroblasts, in a preferred embodiment B16F10, B6, C3, EL4, RMA or RMA-Scells. The cells of the invention which present a polypeptide containinga T-cell epitope may be employed as target cells for restimulatingimmune cells, in particular T cells, and/or for measuring T-cellactivation. A target cell means in accordance with the present inventiona cell which presents a T-cell epitope via MHC molecules and thusspecifically causes T-cell activation, in particular a cytotoxic T-cellreaction against the cell.

Furthermore, the T-cell epitope-containing compound may be part of acomplex which is characterized by the compound being linked covalentlyor by hydrophobic interactions, ionic binding or hydrogen bonds to atleast one further species such as peptides, proteins, peptoids, linearor branched oligo or polysaccharides and nucleic acids.

The present invention therefore relates to a complex containing a T-cellepitope or a compound and at least one further compound. In a preferredembodiment, the polypeptide is linked to MHC class I molecules,preferably as H2-D b tetramer. Particular preference is given to humanor murine MHC class I molecules, in particular an MHC class I moleculederived from C57B1/6 mice. Using the technique by Altman J. D. et al.(1996, Science 274, 94-6) it is possible, for example, to prepareH2-D^(b) tetramers with the appropriate bound peptides which are capableof binding to T-cell receptors of peptide-specific cytotoxic T cells.

Another embodiment is immobilization of the compound of the invention orof said complex to support materials. Examples of suitable supportmaterials are ceramic, metal, in particular noble metal, glasses,plastics, crystalline materials or thin layers of this support, inparticular of said materials, or (bio)molecular filaments such ascellulose or structural proteins.

In order to purify the complex of the invention, a component of thecomplex may additionally also contain a protein tag. Protein tags of theinvention allow, for example, high-affinity absorption to a matrix,stringent washing with suitable buffers with negligible elution of thecomplex and subsequent specific elution of the absorbed complex.Examples of protein tags known to the skilled worker are an N- orC-terminal (HIS)₆ tag, a myc tag, a FLAG tag, a hemagglutinin tag,glutathione transferase (GST) tag, intein with chitin-binding affinitytag or maltose-binding protein (MBP) tag. The protein tags of theinvention may be located N-terminally, C-terminally and/or internally.

The present invention also relates to a method for in vitro detection ofthe activation of T cells by at least one compound containing a T-cellepitope. A method of this kind preferably comprises three steps:

-   -   a) In a first step, cells are stimulated by at least one        compounds containing a T-cell epitope. This compound may be at        least one inventive compound containing a T-cell epitope, at        least one inventive complex containing a T-cell epitope, at        least one capsomer, at least one stable capsomer, at least one        VLP, at least one CVLP, and/or at least one virus. In a        preferred embodiment, immune cells are stimulated by incubation        with CVLPs. This stimulation may be carried out, for example, in        the form of a vaccination or by incubating immune cells with        CVLPs in vitro. Immune cells stimulated in this way are        obtained, for example, after a vaccination from the spleen, from        lymph nodes or from the blood, and/or are cultured.    -   b) In a second step, the cells are incubated with at least one        T-cell epitope of the invention, at least one inventive compound        containing a T-cell epitope, at least one target cell presenting        a T-cell epitope and/or with at least one complex of the        invention.    -   c) In a third step, T-cell activation is determined. Examples of        methods suitable for this are detection of cytokine production        or secretion by the T cells, of the surface molecule expression        on T cells, of target cell lysis or of cell proliferation.        Examples of methods suitable for this are a cytokinassay        (Chapter 6.2 to 6.24 in Current Protocols in Immunology (1999),        edited by Coligan J. E., Kruisbeek A. M., Margulies D. H.,        Shevach E. M. and Strober W., John Wiley & Sons), ELISPOT        (Chapter 6.19 in Current Protocols in Immunology, supra), a ⁵¹Cr        release assay (Chapter 3.11 in Current Protocols in Immunology,        supra) or detection of proliferation (Chapter 3.12 in Current        Protocols in Immunology, supra). Depending on the method used,        it is in this connection also possible to distinguish between        the immune cells such as cytotoxic T cells, T helper cells, B        cells, NK cells, and other cells. The use of inventive        compounds, complexes, and/or cells containing the labels of the        invention allows detection of T cells recognizing the T-cell        epitope via detection of the binding of labeled compounds,        complexes and/or cells to the T-cells. In a preferred        embodiment, binding of inventive MHC-polypeptide complexes to        the surface of T cells is detected. This may be carried out such        that the MHC complexes are labeled themselves, for example        fluorescently labeled, or that, in a further step, an        MHC-specific, labeled, for example fluorescently labeled,        antibody is used in order to detect in turn the MHC complexes.        The fluorescent label of the T cells can then be measured and        evaluated, for example, in a fluorescence-activated cell sorter        (FACS). Another possible way of detecting binding of the        complexes to the T cells is again measuring T-cell activation        (cytokine assay, Elispot, ⁵¹Cr release assay, proliferation, see        above). However, this requires simultaneous stimulation of        coreceptors (e.g. CD28), for example by coreceptor-specific        antibodies (anti-CD28) and/or other unspecific activators        (IL-2).

The present invention also relates to a method containing an additionalstep a′) which is introduced after step a).

-   -   a′) In this additional step a′) which follows step a), the        isolated or cultured cells are are cocultured with at least one        target cell loaded with an inventive compound containing a        T-cell epitope, at least one inventive complex containing a        T-cell epitope, at least one capsomer, at least one stable        capsomer, at least one VLP, at least one CVLP and/or at least        one virus, with at least one inventive complex containing a        T-cell epitope, and/or at least one target cell presenting a        T-cell epitope for at least approx. 12 days, in particular for        approx. 5 days, prior to step b).

Coculturing means growing cells:

-   -   (i) in the presence of at least one target cell loaded with an        inventive compound containing a T-cell epitope, at least one        inventive complex containing a T-cell epitope, at least one        capsomer, at least one stable capsomer, at least one VLP, at        least one CVLP, and/or at least one virus,    -   (ii) in the presence of at least one inventive complex        containing a T-cell epitope,    -   (iii) in the presence of at least one target cell presenting a        T-cell epitope,    -   in the same growth medium and the same tissue culture container.

The present invention further relates to a method for preparing a targetcell presenting a T-cell epitope. It is possible here to load the targetcell with combinations of different T-cell epitopes. In a preferredembodiment, the target cell is incubated with at least one compoundcontaining a T-cell epitope and/or at least one complex containing aT-cell epitope. In a particularly preferred embodiment, the target cellis incubated in growth medium containing polypeptides of the inventionor with MHC class I complexes with bound polypeptides of the invention.The MHC class I complexes may be present for example as H2-D^(b)tetramers. In this connection, a tetramer normally binds four peptides.These can be identical or else represent different peptide species. In afurther preferred embodiment, the target cell is transfected,transformed and/or infected with a nucleic acid and/or a vector. In aparticularly preferred embodiment, the target cell is infected with avaccinia virus vector. The method of the invention is carried out usingantigen-presenting cells, for example B cells, macrophages, dendriticcells, embryonal cells or fibroblasts, and, in a preferred embodiment,using B16F10, B6, C3, EL4, RMA or RMA-S cells.

The CVLPs used contain a papillomavirus L1 protein or variants thereof,in particular HPV16 L1 protein and, but not necessarily, a proteinheterologous to an L1 or variants thereof. The two proteins may be bounddirectly or indirectly. In accordance with the invention, directly boundmeans that the two proteins are covalently bound to one another, forexample via a peptide bond or a disulfide bond. Indirectly bound meansthat the proteins are bound via noncovalent bonds, for examplehydrophobic interactions, ionic bonds or hydrogen bonds. In a furtherembodiment, the CVLPs contain, in addition to L1 protein or variantsthereof, a papillomavirus L2 protein.

Examples of a preferred embodiment of the L1 protein of the presentinvention are L1 proteins having one or more deletions, in particular aC-terminal deletion. A C-terminal deletion has the advantage that it ispossible to increase the efficiency of virus-like particle formation,since the nuclear localization signal located at the C terminus isdeleted. The C-terminal deletion is therefore preferably up to approx.35 amino acids, in particular approx. 25 to approx. 35 amino acids,especially approx. 32 to approx. 34 amino acids. For example, a 32 aminoacid long C-terminal deletion of the HPV16 L1 protein is sufficient inorder to be able to increase the formation of virus-like particles atleast approx. ten times. Furthermore, the L1 protein may carry one ormore mutations or the L1 portion may be composed of L1 proteins ofvarious papillomaviruses. A common characteristic of the L1 proteins ofthe invention is the fact that they permit the formation of VLPs orCVLPs and that they contain at least one T-cell epitope of theinvention.

In a preferred embodiment, the L1 protein or variants thereof and theprotein heterologous to L1 are a fusion protein. Heterologous proteinswhich are composed of a plurality of various proteins or parts thereofare also included. These may also be, for example, epitopes, inparticular cytotoxic T-cell epitopes, of proteins. In this connection,epitopes in accordance with the invention may also be part of asynthetic polypeptide of approx. 50 amino acids, preferably of at leastapprox. 35 amino acids, in particular of at least approx. 20 amino acidsand particularly preferably of at least approx. 9 amino acids, inlength.

Preference is given to proteins heterologous to L1, which are derivedfrom a viral protein, for example derived from HIV, HBV or HCV,preferably from papillomaviruses, in particular from humanpapillomaviruses.

In a preferred embodiment, said viral protein is a papillomavirus Eprotein, preferably an E6 and/or E7 protein. It is particularlypreferred if the E protein is a deleted E protein, preferably aC-terminally deleted, in particular a C-terminally deleted E7 protein,since these constructs in connection with deleted L1 protein can formpreferably virus-like particles. Particular preference is given todeletions of up to 55 amino acids, preferably approx. 5 to approx. 55amino acids, in particular approx. 38 to approx. 55 amino acids.

In a further embodiment, the protein heterologous to L1 may originatefrom antigens of nonviral pathogens. Likewise, they may be derived fromautoimmune antigens such as, for example, thyroglobulin, myelin basicprotein or zona pellucida glycoprotein 3 (ZP₃), which are associatedwith particular autoimmune diseases such as, for example, thyroiditis,multiple sclerosis, oophoritis or rheumatoid arthritis. In a preferredembodiment, the protein heterologous to L1 originates from tumorantigens, preferably melanoma antigens such as MART, ovarian carcinomaantigens such as Her2 neu (c-erbB2), BCRA-1 or CA125, colon carcinomaantigens such as CA125 or breast carcinoma antigens such as Her2 neu(c-erbB2), BCRA-1, BCRA-2.

The present invention further relates to a method for in vitro detectionof the activation of T cells which are obtained by preparation fromsamples. This method makes it possible to determine if a sample, forexample a blood sample of a patient, or murine pancreas containpapillomavirus L1-protein-specific cytotoxic T cells. A detection methodof this kind comprises the following steps:

-   -   a″) In a first step, cells are obtained, for example by taking        blood from a patient or by preparation, for example, of murine        pancreas or lymph nodes. Subsequently, the cells are taken up in        growth medium and cultured.    -   b) In a second step, cells are incubated with at least one        target cell presenting a T-cell epitope or with at least one        complex which comprises as a component a compound containing a        T-cell epitope.    -   c) In a third step, T-cell activation is determined. Examples of        methods suitable for this are detection of cytokine production        or secretion by the T cells, of the surface molecule expression        on T cells, of target cell lysis or of cell proliferation.        Examples of methods suitable for this are a cytokinassay        (Chapter 6.2 to 6.24 in Current Protocols in Immunology (1999),        edited by Coligan J. E., Kruisbeek A. M., Margulies D. H.,        Shevach E. M. and Strober W., John Wiley & Sons), ELISPOT        (Chapter 6.19 in Current Protocols in Immunology, supra), a ⁵¹Cr        release assay (Chapter 3.11 in Current Protocols in Immunology,        supra) or detection of proliferation (Chapter 3.12 in Current        Protocols in Immunology, supra). Depending on the method used,        it is in this connection also possible to distinguish between        the immune cells such as cytotoxic T cells, T helper cells, B        cells, NK cells, and other cells. The use of inventive        compounds, complexes, and/or cells containing the labels of the        invention allows detection of T cells recognizing the T-cell        epitope via detection of the binding of labeled compounds,        complexes and/or cells to the T cells. In a preferred        embodiment, binding of inventive MHC-polypeptide complexes to        the surface of T cells is detected. This may be carried out such        that the MHC complexes are labeled themselves, for example        fluorescently labeled, or that, in a further step, an        MHC-specific, labeled, for example fluorescently labeled,        antibody is used in order to detect in turn the MHC complexes.        The fluorescent label of the T cells can then be measured and        evaluated, for example, in a fluorescence-activated cell sorter        (FACS). Another possibile way of detecting binding of the        complexes to the T cells is again measuring T-cell activation        (cytokine assay, Elispot, ⁵¹Cr release assay, proliferation, see        above). However, this requires simultaneous stimulation of        coreceptors (e.g. CD28), for example by coreceptor-specific        antibodies (anti-CD28) and/or other unspecific activators        (IL-2).

The present invention also relates to a method containing an additionalstep a′) which is introduced after step a″).

-   -   a′) In this additional step a′) which follows step a″), the        isolated or cultured cells are cocultured with at least one        target cell loaded with an inventive compound containing a        T-cell epitope, at least one inventive complex containing a        T-cell epitope, at least one capsomer, at least one stable        capsomer, at least one VLP, at least one CVLP and/or at least        one virus, with at least one inventive complex containing a        T-cell epitope, and/or at least one target cell presenting a        T-cell epitope for at least approx. 12 days, in particular for        approx. 5 days, prior to step b).

Coculturing means growing cells:

-   -   (i) in the presence of at least one target cell loaded with an        inventive compound containing a T-cell epitope, at least one        inventive complex containing a T-cell epitope, at least one        capsomer, at least one stable capsomer, at least one VLP, at        least one CVLP, and/or at least one virus,    -   (ii) in the presence of at least one inventive complex        containing a T-cell epitope,    -   (iii) in the presence of at least one target cell presenting a        T-cell epitope,    -   in the same growth medium and the same tissue culture container.

The invention further relates to an assay system (kit) for in vitrodetection of the activation of T cells, comprising:

-   -   a) at least one T-cell epitope of the invention, at least one        compound of the invention, at least one vector of the invention,        at least one cell of the invention, and/or at least one complex        of the invention, and    -   b) effector cells of the immune system, preferably T cells, in        particular cytotoxic T cells or T helper cells.

The invention further relates to an assay system (kit) for in vitrodetection of the activation of T cells, comprising:

-   -   a) at least one T-cell epitope of the invention, at least one        compound of the invention, at least one vector of the invention,        at least one cell of the invention, and/or at least one complex        of the invention, and    -   b) effector cells of the immune system, preferably T cells, in        particular cytotoxic T cells or T helper cells.

In a particular embodiment, the assay system is used for determining theL1 protein-specific cytotoxic T cells which are present, for example, ina patient's blood sample or in murine pancreas. In this case, the cellsdescribed in b) are control cells contained in the assay system, whoseactivation by the first kit component, the substances mentioned undera), serves as a standard. The activation observed in this reaction iscompared with the T-cell activation of cells, which have been isolatedfrom patients or mice, by kit component a).

In a further particular embodiment, the assay system is used, forexample, for determining the L1 protein-specific antigenicity of acompound containing a T-cell epitope, a complex containing a T-cellepitope, a capsomer, a stable capsomer, a VLP, a CVLP and/or a virus. Inthis case, the substances described in a) are control substances whoseactivating effect on the second kit component, the cells mentioned underb), serves as a standard. The activation observed in this reaction iscompared with the activating effect of a compound containing a T-cellepitope, a complex comprising a T-cell epitope, a capsomer, a stablecapsomer, a VLP, a CVLP, and/or a virus on kit component b).

The invention further relates to the use of at least one T-cell epitope,at least one inventive compound containing a T-cell epitope, at leastone inventive vector containing a nucleic acid coding for a T-cellepitope-containing compound, at least one inventive cell containing aT-cell epitope for, and/or at least one inventive complex containing aT-cell epitope for causing or detecting an immune response.

Suitable cells for immune cell stimulation in vitro as well as in vivoare in particular cells which present at least one of the molecules ofthe invention via their MHC class I molecules. Examples of cellssuitable for antigen presentation are B cells, dendritic cells,macrophages, embryonic cells or fibroblasts which, by being culturedtogether with immune cells, can stimulate specific T cells.

In a particular embodiment, it is possible to use a compound of theinvention, for example an HPV18 L1E7 fusion protein which additionallycontains a T-cell epitope of the invention, for detecting an immuneresponse. Such a compound of the invention may have the ability to formCVLPS.

The invention further relates to a medicament or diagnostic agent whichcontains at least one inventive compound containing a T-cell epitope, atleast one vector containing a nucleic acid coding for a T-cellepitope-containing compound, at least one inventive cell containing aT-cell epitope, and/or at least one inventive complex containing aT-cell epitope and, if necessary, a pharmaceutically acceptable carrier.

Examples of carriers known to the skilled worker are glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylase, natural ormodified cellulose, polyacrylamides, agarose, aluminum hydroxide ormagnetite.

A medicament or diagnostic agent of the invention may be present insolution, bound to a solid matrix, and/or mixed with an adjuvant.

The medicament or diagnostic agent may be administered in differentways. Examples of administration forms known to the skilled worker areparenteral, local and/or systemic administration by, for example, oral,intranasal, intravenous, intramuscular, and/or topical administration.The preferred administration form is influenced, for example, by thenatural path of infection of the particular papillomavirus infection.The amount administered depends on the age, weight and general state ofhealth of the patient and the type of papillomavirus infection. Themedicament or diagnostic agent may be administered in the form ofcapsules, a solution, suspension, elixir (for oral administration) orsterile solutions or suspensions (for parenteral or intranasaladministration). An inert and immunologically acceptable carrier whichmay be used is, for example, a saline or phosphate-buffered saline. Themedicament is administered in therapeutically effective amounts. Theseare amounts which are sufficient for causing a protective immunologicalresponse.

In a particular embodiment, it is possible to use a compound of theinvention, for example an HPV18 L1E7 fusion protein which additionallycontains a T-cell epitope of the invention, as medicament or diagnosticagent. Such a compound of the invention may have the ability to formCVLPs.

The figures and the following examples are intended to illustrate theinvention in more detail, without restricting it.

FIG. 1 shows the graphical analysis of the specific lysis of variousmurine B6 cells which had been infected with either MVA-L1_(Δc) orMVA-F6 by spleen cells of mice which had been vaccinated either withVLPs or with buffer. The percentage of specific lysed cells is plottedas a function of the ratio of effector cells to target cells.

FIG. 2 shows the graphical analysis of the specific lysis of variousRMA-S-cells which present either L1₃₃₀₋₃₃₈, L1₁₆₅₋₁₇₃ or AM by spleencells of mice which had been vaccinated with VLPs or with buffer. Thepercentage of specific lysed cells is plotted as a function of the ratioof effector cells to target cells.

FIG. 3 shows the graphical analysis of the specific lysis of fourdifferent cell lines by T cells which had been restimulated beforehandwith MVA-L1_(Δ)c-infected EL4 cells (left) or with RMA-S-cells loadedwith L1₁₆₅₋₁₇₃. The percentage of specific lysed cells is plotted as afunction of the ratio of effector cells to target cells.

FIG. 4 shows the graphical analysis of the specific lysis of fourdifferent target cell lines by three different cytotoxic T-cell lines.

FIG. 5 shows the graphical analysis of the specific lysis of differenttarget cells, either various cell lines or RMA-S-cells which had beenloaded with L1 peptides L1-1 to L1-15, by three different cytotoxicT-cell lines.

EXAMPLES

1. Description of Starting Materials

-   -   The preparation of HPV16 L1_(Δc)E7₁₋₅₅ CVLPs was carried out        according to the German patent application DE 198 12 941.6, (see        also Muller M. et al. (1997) Virology 234, 93-111).    -   L1 VLPs (see Muller M. et al. (1997) Virology 234, 93-111)    -   C57B1/6 mice were obtained from Charles River Laboratories        (Wilmington, Mass., USA).    -   MVA-L1_(Δc) means a recombinant murine vaccinia virus expressing        HPV16L1_(Δc) in infected cells.    -   MVA-F6 is a vaccinia virus (control virus).    -   B6 cells means embryonic stem cells from a C57B1/6 mouse.    -   C3 cells means HPV16 and ras-transformed B6 embryonic cells (see        Feltkamp M. C. et al. (1993) Eur. J. Immunol. 23, 2242-9).    -   RMA cells originate from a thymoma of a C57BL/6 mouse (see        Ljunggren H. G. & Karre K. (1985) J. Exp. Med. 162, 1745-59).    -   RMA-S cells originate from a thymoma of a C57BL/6 mouse (see        Ljunggren H. G. & Karre K. (1985) J. Exp. Med. 162, 1745-59).        They have a defect in antigen processing-associated transport,        which stops the loading of MHC-1 molecules in the endoplasmic        reticulum. The unloaded MHC-1 molecules which are nevertheless        present on the cell surface may be loaded, for example, by        incubating the cells in peptide-containing media so that these        cells are very suitable for presenting an antigen (see        Powis S. J. et al. (1991) Nature 354, 528-31).    -   EL4 cells originate from a thymoma of a C57B1/6 mouse (see        Shevach E. M. et al. (1972) J. Immunol. 108, 1146-51), for        example ATCC TIB-49.    -   Cells were cultured in each case at 37° C. and 5% CO₂ in RPMI        medium (Gibco BRL, Eggenstein Germany) with 10% fetal calf        serum, kanamycin and ampicillin.    -   AM peptide means amino acids 366 to 374 of influenza        nucleoprotein, sequence: ASNENMETM (see Townsend A. R. et        al. (1986) Cell 44, =959-68) (SEQ ID NO: 3).    -   L1 peptides means HPV16-derived peptide of L1 main capsid        protein.        2. Induction of L1-Specific CTL After Immunization With L1 VLPs        a) Immunization of Mice With VLPS:    -   Two C57B1/6 mice were immunized with 10 μg of L1 VLPs (mouse 1)        or, as a control, with buffer (mouse 2). After 6 weeks, spleen        cells were isolated.        b) Preparation of Antigen-Presenting Cells (Target Cells):    -   B6 cells were incubated with y interferon for 2.5 days, then        infected with MVA-L1_(Δ)c viruses (MOI=5) for preparing        antigen-presenting cells or with MVA-F6 for preparing control        cells overnight and cultured for 16h. In a next step, the        infected B6 cells were irradiated and thus prevented from        further growth.        c) Restimulation of Isolated Spleen Cells:    -   The spleen cells from mouse 1 and 2 were in each case cultured        together with the L1_(Δc)-expressing B6 cells which act as        stimulator cells for T cells of the spleen cells for 5 days.        d) Cytotoxicity Assay of the Isolated Spleen Cells:    -   The stimulated spleen cells (effector cells) of mouse 1        (VLP-vaccinated) and 2 (buffer-vaccinated) were incubated either        with MVA-L1_(Δ)c-infected or with MVA-F6-infected B6 cells        (target cells) which had been incubated in each case beforehand        in the presence of ⁵¹Cr at 37° C. for 1 h, in five different        ratios of effector cells to target cells for 4 h. The specific        lysis of the target cells was measured in a β counter by the        release of radioactive ⁵¹Cr (see FIG. 1).        Result: Spleen cells of the L1-immunized mouse lysed the        MVA-L1_(Δc)-infected target cells, but not the MVA-F6-infected        target cells. The said spleen cells thus had specific        cytotoxicity for the L1 protein. Control spleen cells        originating from the buffer-vaccinated mouse showed no lytic        activity towards the target cells.        3. Peptide-Specific Lysis of Target Cells by L1-Specific T Cells        (VLP-Vaccinated Mice)        a) Preparation of Antigen-Presenting Cells:    -   During a one-hour incubation with ⁵¹Cr at 37° C., RMA-S cells        were additionally incubated with a peptide (concentration 50        μM). The peptide used was either    -   L1_(330-338,)    -   L1₁₆₅₋₁₇₃ or    -   the AM peptide (as a control).        b) Cytotoxicity Assay of the Isolated Spleen Cells:    -   The stimulated spleen cells (effector cells) of mouse 1        (VLP-vaccinated) and 2 (buffer-vaccinated), prepared according        to Example 1c, were incubated with the peptide-incubated RMA-S        cells (target cells) in the presence of 0.5 μg/ml of the        particular peptide in six different ratios of effector cells to        target cells for 4 h. Specific lysis of the target cells was        measured in a counter by the release of radioactive ⁵¹Cr (see        FIG. 2).        Result: It was possible for the spleen cells of L1-immunized        mouse 1 to effectively lyse the L1₁₆₅₋₁₇₃-preincubated target        cells, whereas preincubation of RMA-S cells with L1₃₃₀₋₃₃₈ or        the AM control peptide did not cause any distinct lysis of RMA-S        cells by said stimulated spleen cells. The spleen cells of        buffer-vaccinated mouse 2 showed no specific lytic activity for        any of the peptide-preincubated RMA-S cells. Peptide L1₁₆₅₋₁₇₃        thus represents a specific cytotoxic T-cell epitope in said        cells.        4. Peptide-Specific Lysis of Target Cells by L1-Specific T Cells        (CVLP-Vaccinated Mouse)        a) Immunization of Mice With Cvlps:    -   A mouse was immunized with 10 μg of L1_(Δc)E7₁₋₅₅ CVLPs. Spleen        cells were isolated after 2 weeks.        b) Preparation of Antigen-Presenting Cells:    -   EL4 cells were infected with MVA-L1_(Δc), as described in 1b).        RMA-S cells were loaded with L1₁₆₅₋₁₇₃, as described in 3a).        c) Restimulation of Isolated Spleen Cells:    -   The isolated spleen cells of 4a) were incubated with the two        cell types EL4/MVA-L1_(Δc) and RMA-S/L1₁₆₅₋₁₇₃ described under        4b) in each case for 12 days.        d) Cytotoxicity Assay of the Isolated Spleen Cells    -   Subsequently, a cytotoxicity assay was carried out in analogy to        Example 2d) or 3b). In each case, lysis of EL4/MVA-L1_(Δc) cells        or RMA-S/L1₁₆₅₋₁₇₃ cells was determined in five different ratios        of effector cells to target cells. The controls used were either        EL4 cells which had been infected with an empty MVA-F6 vector,        or RMA-S cells which had not been preincubated with L1₁₆₅₋₁₇₃        (see FIG. 3).        Result: FIG. 3 shows the analysis of the ⁵¹Cr release assay in        analogy to Example 1 or 2. The spleen cells, both after        culturing together with EL4/MVA-L1_(Δc) cells and after        culturing together with RMA-S/L1₁₆₅₋₁₇₃ cells, were capable of        effectively lysing the L1 peptide-presenting target cells        (EL4/MVA-L1_(Δc) cells or RMA-S/L1₁₆₅₋₁₇₃ cells), while the        control cells were not lysed. Since recognition of L1-expressing        cells is comparable to that of peptide-presenting cells, it can        be concluded that peptide L1₁₆₅₋₁₇₃ is essentially responsible        for inducing T-cell response.

This result again indicates that a CD8 T-cell response against HPV16peptide L1₁₆₅₋₁₇₃ is induced in CVLP-vaccinated mice. In thisconnection, it is unimportant whether vaccination of the mice wascarried out using VLPs (see Example 3) or CVLPs (this example).

5. L1-Specific Cytotoxic T Cells

A mouse was vaccinated twice with 10⁷ C3 cells (HPV16- andras-transformed). The spleen cells were subsequently isolated andcultured in the presence of irradiated C3 cells as stimulator cells andrestimulated by the addition of C3 cells. “Spleen cell clones” werecultured by thinning out the spleen cells. Said clones were then assayedin cytotoxicity assays on how efficiently they lyse C3 cells. In thesame assay, said clones lysed substantially less efficiently B6, RMA orRMA-E7 cells (due to transformation with HPV16, C3 cells express E7,too). FIG. 4 shows three of these cytotoxic clones (=T-cell lines) whichlyse C3 cells distinctly more efficiently than B6, RMA-E7 or RMA cells.

Said three T-cell lines were then assayed in a cytotoxicity assayaccording to any of the preceding examples for their capability oflysing C3 cells, B6 cells, RMA-S cells and also RMA-S cells which hadbeen loaded beforehand with various L1 peptides (L1-1 to L1-15;concentration in each case 50 μM). FIG. 5 shows that T-cell lines 7A and11C are able to lyse very efficiently L1-14-loaded RMA-S cells. Thus,said T-cell lines are specific for peptide L1-14 which is L1 peptide330-338 (L1₃₃₀₋₃₃₈). The other assayed peptides were not recognized bysaid T-cell lines. The sequences of said assayed peptides are asfollows: L1-1: GAMDFTTL (SEQ ID NO: 4), L1-2: GDSLFFYL (SEQ ID NO: 5),L1-3: MQVTFIYI (SEQ ID NO: 6), L1-4: VYHIFFQM (SEQ ID NO: 7), L1-5.VHTGFGAM (SEQ ID NO: 8), L1-6: KYPDYIKM (SEQ ID NO: 9), L1-7: VTFIYILV(SEQ ID NO: 10), L1-8: LEDTYRFV (SEQ ID NO: 11), L1-9: GNQLFVTV (SEQ IDNO: 12), L1-10: KKYTFVTV (SEQ ID NO: 13), L1-11: ENDVNYHI (SEQ ID NO:14), L1-12: AGVDNRECI (SEQ ID NO: 15), L1-13: TVGENVPDDL (SEQ ID NO:16), L1-14: AQIFNKPYW (SEQ ID NO: 1), L1-15: YKNTNFKEYL (SEQ ID NO: 17)

1. A T-cell epitope consisting of an amino acid sequence selected fromthe group consisting of AQIFNKPYW (SEQ ID NO: 1) and AGVDNRECI (SEQ IDNO: 2).